Treatments for cancer

ABSTRACT

The present invention provides methods for reducing tumor survival, expansion, and metastasis. In particular, the invention provides methods for reducing pancreatic tumor survival, expansion, and metastasis. The invention also provides agents for use in the methods, particularly agents that reduce the level or activity of connective tissue growth factor (CTGF), and methods for identifying such agents.

This application is a continuation of U.S. application Ser. No.11/119,309, filed 28 Apr. 2005, which claims the benefit of U.S.Provisional Application No. 60/588,843, filed 16 Jul. 2004 and U.S.Provisional Application Ser. No. 60/566,277, filed on 28 Apr. 2004, bothof which are incorporated in their entirety by reference herein.

FIELD OF THE INVENTION

The present invention provides methods for reducing tumor survival,expansion, and metastasis. In particular, the invention provides methodsfor reducing pancreatic tumor survival, expansion, and metastasis. Theinvention also provides agents for use in the methods, particularlyagents that reduce the level or activity of connective tissue growthfactor (CTGF), and methods for identifying such agents.

BACKGROUND OF THE INVENTION

Cancer affects the lives of millions of people on a global basis.Treatments such as chemotherapy produce beneficial results in somemalignancies, however, some cancers, including lung, pancreatic,prostate, and colon cancers, demonstrate poor response to suchtreatments. Further, even cancers initially responsive to chemotherapycan return after remission, with widespread metastatic spread leading todeath of the patient. In addition, chemotherapy agents, e.g.,antineoplastic agents, have significant toxicities, and are associatedwith side effects including, e.g., bone marrow suppression, renaldysfunction, stomatitis, enteritis, and hair loss. Therefore, there is aneed for effective and safe therapies for treatment of cancer,prevention of metastasis, etc.

Connective Tissue Growth Factor (CTGF) is a growth factor withdemonstrated effects in various physiological and pathological contexts,including mitogenic and chemotactic processes, and the production ofextracellular matrix components. CTGF has been implicated in a number ofdisorders and conditions, including, but not limited to, disordersinvolving angiogenesis, fibrosis, and other conditions withproliferative aspects. CTGF has been previously identified as a criticalfactor associated with tumor formation and growth, and is overexpressedin a variety of tumor types. (See, e.g., International Publication No.WO 96/38172; Wenger et al. (1999) Oncogene 18:1073-1080; Xie et al.(2001) Cancer Res 61:8917-8923; Igarashi et al. (1998) J Cutan Pathol25:143-148; Kasaragod et al. (2001) Ped Dev Pathol 4:3745; Shakunaga etal. (2000) Cancer 89:1466-1473; Vorwerk et al. (2000) Br J Cancer83:756-760; Pan et al. (2002) Neurol Res 24(7):677-683.) CTGF is alsoknown to have pro-angiogenic activity in vivo, an important processassociated with tumor survival. (See, e.g., Brigstock (2002)Angiogenesis 5:153-165; Shimo et al. (1999) J Biochem 126:137-145; Babicet al. (1999) Mol Cell Biol 19:2958-2966; Ivkovic et al. (2003)Development 130:2779-2791; and Shimo et al. (2001) Oncology 61:315-322.)

However, correlation between CTGF expression and prognosis in cancerpatients has suggested a context specific role for the protein. Forexample, CTGF expression was associated with longer patient survival insquamous cell carcinomas, but decreased survival in esophagealadenocarcinomas. (Koliopanos et al. (2002) World J Surg 26:420-427.)Similarly, CTGF has been implicated in increased apoptosis of, e.g.,breast cancer cells, and increased survival of, e.g., rhabdomyosarcomacells. (See, e.g., Hishikawa et al. (1999) J Biol Chem 274:37461-37466;Croci et al. (2004) Cancer Res 64:1730-1736.) Therefore, there is a needfor improved understanding of cancer-associated CTGF-related effects,and for methodologies appropriately targeting CTGF within the context ofthe disease.

In summary, there is a need in the art for effective treatments forcancer, and, specifically, there is a need for methods of selectivetreatment that effectively targets CTGF-related or -induced aspects ofcancer. The present invention meets these needs by providing methods forreducing tumor survival, expansion, and metastasis, and, in particular,by providing methods for reducing pancreatic tumor survival, expansion,and metastasis. The invention also provides agents for use in themethods, particularly reagents that reduce the level or activity ofCTGF, and methods for identifying such agents.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method foridentifying an agent that inhibits anchorage-independent cell growth,the method comprising (a) culturing MIA PaCa-2 cells, wherein the cellshave been modified to express connective tissue growth factor (CTGF),with an agent under conditions suitable, in the absence of the agent,for anchorage-independent cell growth; (b) measuring the amount of cellgrowth that occurs in the presence of the agent; and (c) comparing theamount of cell growth that occurs in the presence of the agent with theamount of cell growth that occurs in the absence of the agent, wherein adecrease in the amount of cell growth in the presence of the agentrelative to the amount of cell growth that occurs in the absence of theagent is indicative of an agent that inhibits anchorage-independent cellgrowth. In a certain embodiment, the CTGF is human CTGF. In anotherembodiment, the MIA PaCa-2 cells, when 10⁵ cells are cultured in asuitable growth medium, secrete into the culture medium at least about0.3 μg CTGF/48 hrs.

In some embodiments, the measuring the amount of cell growth comprisescounting clusters of cells. In a further embodiment, the culturingcomprises dispersing the cells in a semi-solid support mediumappropriate for anchorage-independent cell growth; and, in a specificembodiment, the semi-solid support medium comprises about 0.35% agar.

The present invention further encompasses various uses for agents thatinhibit anchorage-independent cell growth identified by theabove-described methods. In one aspect, the invention provides a methodfor reducing metastasis of a tumor in a subject, the method comprisingadministering to the subject an agent identified by any one of theabove-described methods for identifying an agent that inhibitsanchorage-independent cell growth, thereby reducing metastasis of thetumor in the subject. In a particular aspect, the tumor is a pancreatictumor.

In another aspect, the invention provides a method for reducingexpansion of a pancreatic tumor in a subject, the method comprisingadministering to the subject an agent identified by any one of theabove-described methods for identifying an agent that inhibitsanchorage-independent cell growth, thereby reducing pancreatic tumorexpansion in the subject. In another aspect, the invention providesmethods for reducing pancreatic tumor cell survival in a subject, themethod comprising administering to the subject an agent identified bythe above-described methods for identifying an agent that inhibitsanchorage-independent cell growth, thereby reducing pancreatic tumorcell survival in the subject.

The invention further encompasses a method for reducing metastasis of atumor in a subject, the method comprising administering to the subjectan agent that inhibits CTGF activity, thereby reducing metastasis of thetumor. In a specific embodiment, the tumor is a pancreatic tumor. Inpreferred embodiments, the subject is a mammal, and, in a most preferredembodiment, a human. Whether an agent inhibits CTGF activity can bedetermined by any one of a number of methods well-known to those in theart, for example, using the anchorage-independent growth assay asdescribed above (supra).

In one embodiment, the tumor is an adenocarcinoma, and, in a furtherembodiment, the tumor is a ductal adenocarcinoma.

In various embodiments, the agent is an oligonucleotide thatspecifically binds to a polynucleotide encoding CTGF; is a smallmolecule; or is an aptamer. In a preferred embodiment, the agent is anantibody that specifically binds to CTGF. In certain embodiments, theantibody that specifically binds to CTGF is a monoclonal antibody or anactive fragment thereof, or is CLN-1 or any derivative thereof. In afurther embodiment, a cytotoxic chemotherapeutic agent is alsoadministered to the subject.

Methods for reducing or inhibiting progression of cancer in a subjectare also contemplated herein, the method comprising administering to thesubject an agent that reduces metastasis of a tumor and that reduces atleast one process selected from the group consisting of tumor expansionand tumor cell survival, thereby reducing or inhibiting the progressionof cancer in the subject. In certain embodiments, the tumor is apancreatic tumor.

A method for reducing metastasis of a pancreatic tumor in a subject, themethod comprising administering to the subject an agent that inhibitsCTGF activity, thereby reducing metastasis, is specifically contemplatedherein. The invention further provides methods for reducing pancreatictumor cell or pancreatic tumor survival in a subject, the methodcomprising administering to the subject an agent that inhibits CTGFactivity, thereby reducing pancreatic tumor cell or pancreatic tumorsurvival. A method for reducing pancreatic tumor expansion in a subject,the method comprising administering to the subject an agent thatinhibits CTGF activity, thereby reducing pancreatic tumor expansion, isalso provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows CTGF expression levels in various MIA PaCa-2 clonal celllines stably transformed with a CTGF-expression construct. Cell linesrepresenting “low” (CB1 and CB2), “medium” (CE8 and CD2), and “high”(CA9 and CB4) CTGF production were used in the examples provided herein.

FIG. 2 shows growth characteristics of MIA PaCa-2 cells stablytransformed with CTGF-encoding expression constructs. FIG. 2A shows thatthe level of CTGF produced by MIA PaCa-2 cells does not affect cellgrowth when cultured on a 2-dimensional surface. FIG. 2B, however, showsthat the level of CTGF produced does affect cell growth and colonyformation when cells are cultured in a soft agar medium.

FIG. 3 shows dependence of anchorage independent growth on CTGF. FIG. 3Ashows images of cells expressing no (vector) or medium levels (med) ofCTGF. Top panels show that CTGF increases the number and appearance ofsoft agar colonies; lower panels show that treatment with an antibodythat binds CTGF substantially reduces both number and size of colonies.FIG. 3B shows a quantitative difference in colony number due to CTGFexpression level and antibody treatment.

FIGS. 4A and 4B show changes in tumor volume and mortality,respectively, in mice bearing tumors derived from the medium- andhigh-CTGF expressing cells.

FIG. 5 shows that cell survival in tumors in situ is correlated withlevel of CTGF. FIG. 5A shows an increase in proliferating cells and FIG.5B shows a decrease in apoptotic cells in tumors derived from CTGFexpressing cells relative to vector control cells.

FIGS. 6A, 6B, and 6C show correspondence between CTGF expression levelin vitro and plasma and urine CTGF levels in tumor-bearing animals forlow-, medium-, and high-CTGF expressing cells and tumors derivedtherefrom.

FIGS. 7A and 7B show that therapeutics targeting CTGF effectively reducesurvival and expansion of tumors derived from MIA PaCa-2 cellsexpressing high levels of CTGF and Panc-1 cells, respectively.

FIG. 8 shows cell survival in tumors in situ is dependent upon CTGF.FIG. 8A shows a therapeutic targeting CTGF does not significantly alterthe proliferative capacity of the cells, and FIG. 8B shows that an agenttargeting CTGF significantly increases apoptosis.

FIG. 9 shows survival, expansion, and metastasis of tumors derived fromorthotopic implantation of PANC-1 cells into the pancreas of mice, andthe ability of an agent targeting CTGF to reduce both survival andmetastases of the tumors.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that the invention is not limited to the particularmethodologies, protocols, cell lines, assays, and reagents described, asthese may vary. It is also to be understood that the terminology usedherein is intended to describe particular embodiments of the presentinvention, and is in no way intended to limit the scope of the presentinvention as set forth in the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural references unlesscontext clearly dictates otherwise. Thus, for example, a reference to “afragment” includes a plurality of such fragments; a reference to an“antibody” is a reference to one or more antibodies and to equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications cited hereinare incorporated herein by reference in their entirety for the purposeof describing and disclosing the methodologies, reagents, and toolsreported in the publications that might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, cell biology, genetics, immunology and pharmacology, within theskill of the art. Such techniques are explained fully in the literature.

Invention

The present invention provides methods and compounds for treating cancerin a subject, in particular, pancreatic cancer, by inhibiting theexpression and/or activity of CTGF. In particular, the inventionprovides methods for reducing tumor metastasis and for reducing tumorsurvival and expansion by inhibiting the expression and/or activity ofCTGF. In certain embodiments, the invention provides methods forreducing pancreatic tumor metastasis, pancreatic tumor survival, andpancreatic tumor expansion. In another embodiment, the methods reduce orinhibit metastasis of tumors to bone.

In one embodiment, the invention provides a method of reducing orinhibiting tumor expansion in a subject, the method comprisingadministering to the subject an effective amount of an agent thatinhibits CTGF expression and/or activity. Tumor expansion may includevarious aspects of tumor biology including, but not limited to,modulation of boundary between the tumor and surrounding stroma;signaling and recruitment of neighboring, non-transformed cells; etc. Ina particular embodiment, the invention provides a method for inhibitingpancreatic tumor expansion in a subject, the method comprisingadministering to the subject an effective amount of an agent thatinhibits CTGF expression and/or activity. In various embodiments, thesubject is a mammal, particularly a human. In various aspects, the agentis an antibody, a small molecule, or an oligonucleotide-based moleculesuch as aptamers and antisense.

In another embodiment, the invention provides a method of reducing orinhibiting tumor survival in a subject, particularly tumor cellsurvival, the method comprising administering to the subject aneffective amount of an agent that inhibits CTGF expression and/oractivity. Tumor cell survival may include various aspects of tumorbiology including, but not limited to, modulating apoptotic potential;etc. In a particular embodiment, the invention provides a method forinhibiting pancreatic tumor survival, particularly pancreatic tumor cellsurvival, in a subject, the method comprising administering to thesubject an effective amount of an agent that inhibits CTGF expressionand/or activity. In various embodiments, the subject is a mammal,particularly a human. In various aspects, the agent is an antibody, asmall molecule, or an oligonucleotide-based molecule such as aptamersand antisense.

In another embodiment, the invention provides a method for reducing orpreventing tumor metastasis in a subject, the method comprisingadministering to the subject an effective amount of an agent thatinhibits CTGF expression or activity. Metastasis may involve and includeprocesses at the site of the primary tumor that facilitate tumorinvasion of neighboring tissues, organs, etc., or invasion of lymphaticand/or circulatory systems. Metastasis may also involve and includeprocesses at the site of a secondary tumor that facilitates attachmentand invasion by the metastasized tumor. Such processes may be due to thecancer cell, e.g., overexpression of CTGF within the cancer cell, etc.;or due to the endogenous tissue at the site of metastasis, e.g., CTGFoverexpression by stromal tissue and/or response of bone, liver, etc. toCTGF. In a particular aspect, the invention provides a method forinhibiting or preventing metastasis of a pancreatic tumor in a subject,the method comprising administering to a subject an effective amount ofan agent that inhibits CTGF expression or activity. In variousembodiments, the subject is a mammal, particularly a human. In variousaspects, the agent is an antibody, a small molecule, or anoligonucleotide-based molecule such as aptamers and antisense.

Methods for reducing or preventing mortality associated with cancer,particularly pancreatic cancer, are also provided, the methodscomprising administering to a subject having cancer or at risk forhaving cancer an effective amount of an agent that inhibits theexpression and/or activity of CTGF. In various aspects, the subject is amammal, particularly a human. In various embodiments, the agent is anantibody, a small molecule, or an oligonucleotide-based molecule such asaptamers and antisense.

The present invention establishes for the first time a direct causalrelationship between CTGF and the survival and expansion of tumors,particularly pancreatic tumors. Pancreatic tumor, as used herein,includes any tumor located in, derived from, or originating from cellsof the pancreas. This includes primary tumors originating in thepancreas, secondary tumors originating in the pancreas or another organ,etc. Further, the present invention demonstrates the explicitcorrelation between CTGF expression and tumor cell survival, tumorexpansion, extent of metastasis, etc. The present invention furtherdemonstrates that agents or compounds that target CTGF, therebypotentially inhibiting the expression and/or activity of CTGF,effectively reduce tumor expansion, increase tumor cell apoptosis,reduce metastasis of tumors, and improve subject survivability. Inparticular, the invention demonstrates that agents or compounds thattarget CTGF effectively reduce pancreatic tumor expansion, increasepancreatic tumor cell apoptosis, and reduce metastasis of pancreatictumors.

Connective Tissue Growth Factor (CTGF)

CTGF is a 36 kD, cysteine-rich, heparin binding, secreted glycoproteinoriginally isolated from the culture media of human umbilical veinendothelial cells. (Bradham et al. (1991) J Cell Biol 114:1285-1294;Grotendorst and Bradham, U.S. Pat. No. 5,408,040.) CTGF belongs to theCCN (CTGF, Cyr61, {umlaut over (N)}ov) family of proteins, whichincludes the serum-induced immediate early gene product Cyr61, theputative oncogene Nov, and the Wnt-inducible secreted proteins (WISP)-1,-2, and -3. (See, e.g., O'Brian et al. (1990) Mol Cell Biol10:3569-3577; Joliot et al. (1992) Mol Cell Biol 12:10-21; Ryseck et al.(1991) Cell Growth and Diff 2:225-233; Simmons et al. (1989) Proc. Natl.Acad. Sci. USA 86:1178-1182; Pennica et al. (1998) Proc Natl Acad SciUSA, 95:14717-14722; and Zhang et al. (1998) Mol Cell Biol18:6131-6141.) CCN proteins are characterized by conservation of 38cysteine residues that constitute over 10% of the total amino acidcontent and give rise to a modular structure with N- and C-terminaldomains. The modular structure of CTGF includes conserved motifs forinsulin-like growth factor binding proteins (IGF-BP) and vonWillebrand's factor (VWC) in the N-terminal domain, and thrombospondin(TSPI) and a cysteine-knot motif in the C-terminal domain.

Although the present invention demonstrates the direct role of CTGF intumor survival, expansion, and metastasis, and demonstrates that agentstargeting CTGF are beneficial in treating cancer, the inventionspecifically contemplates a similar role for other CCN family members,particularly Cyr61.

CTGF expression is induced by various factors including TGF-β familymembers, e.g., TGF-⊕1, activin, etc.; thrombin, vascular endothelialgrowth factor (VEGF), endothelin and angiotensin II. (Franklin (1997)Int J Biochem Cell Biol 29:79-89; Wunderlich (2000) Graefes Arch ClinExp Opthalmol 238:910-915; Denton and Abraham (2001) Curr Opin Rheumatol13:505-511; and Riewald (2001) Blood 97:3109-3116; Xu et al. (2004) JBiol Chem 279:23098-23103.) Such factors have been associated withtumorigenesis previously. Therefore, in one aspect the present inventionis directed to treatment of cancers whose negative prognosis iscorrelated with these factors, e.g., TGF-β.

CTGF has been associated with various neoplasms, but its specific rolehas not been clearly elucidated. CTGF was originally described as amitogenic factor and was linked to tumor cell proliferation, therebyaffecting the formation and growth of the tumor. Expression of CTGFappears to occur in cells both within and bordering the tumor, leadingsome investigators to suggest that CTGF may facilitate reorganization ofthe extracellular matrix and promote neovascularization of the tumor.(See, e.g., Shimo et al. (2001) Oncogene 61(4):315-22; Pan et al. (2002)Neurol Res 24(7):677-83; Kondo et al. (2002) Carcinogenesis23(5):769-76.) CTGF has been implicated in both increased apoptosis,e.g., in breast cancer cells, and increased survival, e.g., inrhabdomyosarcoma cells. (See, e.g., Hishikawa et al. (1999) J Biol Chem274:37461-37466; Croci et al. (2004) Cancer Res 64:1730-1736.)Therefore, the role of CTGF in cancer may be context-specific, dependingon the type and origin of the primary tumor.

CTGF is specifically expressed in malignant lymphoblasts in acutelymphoblastic leukemia (ALL), and CTGF expression is highly correlatedwith tumor stage in breast cancer and glioma. (See, e.g., Xie et al.(2001) Cancer Res 61:8917-8923; and Xie et al. (2004) Clin Cancer Res10:2072-2081.) CTGF expression has also been associated with invasivepancreatic cancer, and in breast cancer that metastasizes to bone. (See,e.g., Iacobuzio-Donahue et al. (2002) Am J Pathol 160:91-99; Kang et al.(2003) Cancer Cell 3:537-549.) In metastatic breast cancer, CTGF isover-expressed in metastatic cells that induce osteolysis, where tumorcell-mediated interaction with and/or degradation of bone matrixreleases growth factors that activate osteoclasts leading to boneresorption. Additional metastastic cancers that exhibit prominentosteolytic phenotypes or that metastasize to bone are prostate,hepatocellular carcinoma, colorectal, pancreatic, ovarian, renal cellcarcinoma, multiple myeloma, lymphoma, and leukemia.

The present invention, for the first time, demonstrates a direct causalrelationship between CTGF and tumor cell survival, tumor expansion, andmetastasis of tumors. For example, the present invention demonstratesthat tumors derived from MIA PaCa-2 cells transfected with a CTGFexpression construct show enhanced tumor cell survival, increased tumorsize and invasiveness, and a greater propensity of primary tumors tometastasize; and that animals bearing such tumors show increasedmortality. The present invention further demonstrates that tumorexpansion, metastasis, and patient mortality correlate with levels ofCTGF expression, i.e., mice implanted with cells expressing high levelsof CTGF display a higher level of tumor expansion and mortality thanmice implanted with cells expressing lower levels of CTGF, e.g.,medium-CTGF expressing cells.

Still further, the present invention demonstrates that in vivoadministration of an agent that inhibits expression or activity of CTGFslows or prevents tumor expansion. In particular, mice bearing tumorsderived from cells expressing medium and high levels of CTGF showreduced tumor expansion and reduced mortality upon treatment with ananti-CTGF antibody. This demonstrates that CTGF expression is causallylinked to tumor survival and expansion, and associated host mortality,and that therapeutics targeting CTGF may be effective at retarding tumorexpansion and reducing mortality in cancer patients.

Although not to be limited by any particular mechanism, the presentinvention contemplates the role of CTGF in regulating the activity ofNFκB, a transcription factor associated with cell proliferation and cellsurvival pathways in various disorders including pancreatic cancer.(See, e.g., Algul (2002) Int J Gastrointest Cancer 31:71-78.) Regulationof NFκB likely involves other components of signaling, including, butnot limited to, inhibition of IκB and modulation of glycogen synthasekinase (GSK)-3β. (See, e.g., Hoeflich et al. (2000) Nature 406:86-90.)In one aspect, the present invention contemplates a signaling pathwaythat involves activation of GSK-3β and NFκB by a CTGF-dependentmechanism, and provides agents that modulate the expression and/oractivity of CTGF to further modulate downstream GSK-3β and NFκBactivity.

Thus, in one aspect, the present methods and compounds are applied totreatment of a wide variety of cancers including, but not limited to,adenocarcinomas, particularly ductal carcinomas as frequently occur inbreast and pancreatic cancer; neuroepithelial tumors, e.g., gliomas;gastrointestinal carcinoids, particularly ileal carcinoids; acutelymphoblastic leukemia, rhabdomyosarcoma, and melanoma; and in cancerswith a propensity for metastasis to bone, particularly wherein thesecondary tumor produces effects on the bone, including osteolytic andosteoblastic lesion formation. In particular aspects, use of the presentmethods and compounds to treat pancreatic cancer is provided.

Screening Assay

In another aspect, the present invention provides methods foridentification of agents or compounds for reducing tumor survival andexpansion, particularly tumors of the pancreas. Methods of identifyingcompounds or agents use various procedures described in the examplesherein; e.g., an animal bearing a tumor derived from a CTGF-expressingcell is treated with a compound or agent and tumor expansion andmetastasis are measured. An agent that retards or prevents tumorexpansion and survival, and/or prevents or reduces tumor metastasiswould be selected for use in the present methods.

In one embodiment, the present invention provides a screening assay foridentifying an agent that modulates anchorage-independent cell growth(AIG), which is a demonstrated characteristic of tumorigenic cells. Themethod comprises culturing a CTGF-expressing cell with an agent underconditions suitable, in the absence of the agent, foranchorage-independent growth of the cell; measuring the amount of cellgrowth that occurs in the presence of the agent; and comparing theamount of cell growth that occurs in the presence of the agent with theamount of cell growth that occurs in the absence of the agent, wherein achange in the amount of cell growth in the presence of the agentrelative to the amount of cell growth that occurs in the absence of theagent is indicative of an agent that modulates AIG.

In certain embodiments, the cells used in the assay express CTGFendogenously, whereas in other embodiments the cells have beenrecombinantly modified to express CTGF. Any cell expressing CTGF can beutilized in the assay. In some embodiments described herein, the cell isa PANC-1 cell. PANC-1 cells are ductal epithelioid carcinoma cellsoriginally obtained from a primary pancreatic cancer. (Lieber et al.(1975) Int J Cancer 15:741-747.) In other embodiments described herein,the cell is a MIA PaCa-2 cell recombinantly modified to express CTGF.MIA PaCa-2 cells were originally obtained from a human primarypancreatic cancer. (Yunis et al. (1975) Int J Cancer 19:128-135.) Whenthe cells have been modified to express CTGF, the CTGF can be anynaturally occurring CTGF protein, e.g., human CTGF, mouse FISP-12, etc;or any synthetic CTGF protein retaining the requisite activity, i.e.,modulation of AIG, of a naturally-occurring CTGF. In a particularembodiment, the CTGF is human CTGF. The cells typically expresssufficient amounts of CTGF to produce measurable activity, e.g., cellsurvival, colony expansion, etc., in a given period of time. Forexample, when approximately 10⁵ cells producing endogenous CTGF orstably transfected with a CTGF expression construct are placed in anappropriate growth medium and cultured for 48 hours, at least 0.3 μgCTGF, and more specifically about 0.3 to 2.2 μg CTGF, will be producedand secreted into the medium.

In particular embodiments of the screening assay, cells expressing CTGFmay be dispersed in a semi-solid support medium appropriate for AIG. Inone embodiment, the semi-solid support medium comprises agar, e.g.,Noble Agar. The agar can be at any suitable concentration to supportAIG; in one particular embodiment, the agar is at a concentration ofabout 0.35%. Cell growth can be measured by any method known to those ofskill in the art. In one embodiment, cell growth comprises countingclusters of cells in a defined area of the semi-solid support medium. Inanother embodiment, cell growth comprises measuring average clustersize.

Compounds and Agents

Various compounds and agents can be used in the present methods. Thecompounds and agents modulate CTGF activity, such activity including,but not limited to, expression of CTGF by a cell and alterations in cellphenotype due to CTGF. Alterations in cell phenotype may includemodification of cell surface or intracellular macromolecules, such asproteins, e.g., by phosphorylation or de-phosphorylation; changes ingene expression as detected, e.g., by microarray or quantitative PCRanalysis; and/or changes in protein expression and/or secretion, e.g.,increased production of collagen by a cell. Alterations in cellphenotype may also include, but are not limited to, gross changes incell shape, adhesion of a cell to another cell and/or to extracellularmatrix, and cell motility as measured, e.g., in a Boyden chamber assay.In particular, compounds and agents for use in the present methodsmodulate anchorage-independent growth of a cell as measured, e.g., in anassay as described above or provided in the examples herein.

In some embodiments, the agent is an antibody that specifically binds toCTGF. In a preferred embodiment, the antibody is a monoclonal antibody.In another preferred embodiment, the antibody is a human or humanizedantibody. Antibodies that bind to CTGF are described in U.S. Pat. No.5,408,040; International Publication No. WO 99/07407; InternationalPublication No. WO 99/33878; and International Publication No. WO00/35936, each of which reference is incorporated herein in itsentirety. In particular embodiments, the agent comprises CLN-1,described in International Publication No. WO 2004/108764, whichreference is incorporated herein in its entirety. Any antibody thatspecifically binds to CTGF, such as any of the above-describedantibodies, may be used in the present methods in its entirety, or maybe modified to obtain a portion of the antibody that retains appropriateactivity against CTGF, e.g., epitope-binding activity, and which portioncan thus be used in the present methods. For example, an antibodyderived from CLN-1 may comprise a CLN-1 light chain, a CLN-1 heavychain, or variable domains of the heavy and light chain. In oneparticular embodiment, the antibody of CLN-1 is the antibody produced byATCC cell line accession no. PTA-6006. Antibodies, or fragments thereof,can be administered by various means known to those skilled in the art.For example, antibodies are often injected intravenously,intraperitoneally, or subcutaneously. Antibody formulations may also beinjected intraarticularly, intraocularly, intradermally, intrathecallyetc.

In other embodiments, the agent is a small molecule. Small moleculeinhibitors of CTGF expression and/or activity have been described; forexample, International Publication No. WO 96/38172 identifies modulatorsof cAMP such as cholera toxin and 8Br-cAMP as inhibitors of CTGFexpression. Therefore, compounds known to increase cAMP level in cells,e.g., prostaglandin and/or prostacyclin analogs such as Iloprost;phosphodiesterase IV inhibitors; etc., may be used to modulate CTGFexpression. (See, e.g., International Publication No. WO 00/02450;Ricupero et al. (1999) Am J Physiol 277:L1165-1171; also, see Ertl etal. (1992) Am Rev Respir Dis 145:A19; and Kohyama et al. (2002) Am JRespir Cell Mol Biol 26:694-701.) Also, inhibitors of serine/threoninemitogen activated kinases, particularly p38, cyclin-dependent kinase,e.g. CDK2, and glycogen synthase kinase (GSK)-3 have also beenimplicated in decreased CTGF expression and/or signaling. (See, e.g.,Matsuoka et al. (2002) Am J Physiol Lung Cell Mol Physiol 283:L103-L112;Yosimichi et al. (2001) Eur J Biochem 268:6058-6065; InternationalPublication No. WO 01/38532; and International Publication No. WO03/092584.) Such agents can be used to reduce expression/activity ofCTGF and thereby ameliorate or prevent the pathological processesinduced by CTGF. Such compounds can be formulated and administeredaccording to established procedures within the art. (See, e.g., Gennaro,Ed. (2000) Remington's Pharmaceutical Sciences, 20^(th) edition, MackPublishing Co., Easton Pa.; and Hardman, Limbird, and Gilman, Eds.(2001) The Pharmacological Basis of Therapeutics, 10^(th) Edition,McGraw Hill Co, New York N.Y.)

In various embodiments, the agent is an antisense or aptamer oligo- orpolynucleotide. Antisense technologies, including small interferingribonucleic acids (siRNAs), micro-RNAs (miRNAs), ribozymes, andantisense sequences directed to modulate CTGF expression may also beused to inhibit tumor expansion and survival, and metastasis, and todecrease associated mortality rates. (See, e.g., Zeng (2003) Proc NatlAcad Sci USA 100:9779-9784; and Kurreck (2003) Eur J Biochem270:1628-1644.) Antisense constructs that target CTGF expression havebeen described and utilized to reduce CTGF expression in various celltypes. (See, e.g., International Publication No. WO 96/38172;International Publication No. WO 00/27868; International Publication No.WO 00/35936; International Publication No. WO 03/053340; Kothapalli etal. (1997) Cell Growth Differ 8(1):61-68; Shimo et al. (1998) J Biochem(Tokyo) 124(1): 130-140; and Uchio et al. (2004) Wound Repair Regen12:60-66.) Such antisense constructs can be used to reduce expression ofCTGF and thereby ameliorate or prevent CTGF-modulated effects, e.g.,tumor progression and/or metastasis, etc. Such constructs can bedesigned using appropriate vectors and expressional regulators for cell-or tissue-specific expression and constitutive or inducible expression.These genetic constructs can be formulated and administered according toestablished procedures within the art.

Aptamers that modulate CTGF activity may be RNA or DNA oligonucleotidesor proteins identified, e.g., using the screening assay provided herein.Aptamers are generally identified through a process called SELEX(systematic evolution of ligands by exponential enrichment), aniterative screening process for selection and amplification of anaptamer having appropriate activity and selectivity. (See, e.g.,Ellington and Szostak (1990) Nature 346:818-822; Klug and Famulok (1994)Mol Biol Rep 20:97-107; and Brody and Gold (2000) J Biotechnol 74:5-13.)Aptamer libraries, typically composed of, e.g., single stranded DNA orRNA oligonucleotides containing a central region of randomized sequencesflanked by constant regions for subsequent transcription, reversetranscription, and DNA amplification, are readily available or prepared.(See, e.g., Fiegon et al. (1996) Chem Biol 3:611-617.)

In some aspects, compound or agent is administered to a subject toreduce or prevent tumor cell survivability, tumor expansion, and tumormetastasis. In certain embodiments, the compound or agent specificallyand selectively targets CTGF, e.g., inhibiting the expression and/oractivity of CTGF with no significant effect on other factors. In otherembodiments, the compound or agent specifically and selectively targetsCCN family members, e.g., inhibiting the expression and/or activity ofCCN family members with no significant effect on other factors. Inparticular aspects, the CCN family members are selected from the groupconsisting of CTGF and Cyr61.

The compound or agent may be administered alone or in combination withone or more additional therapeutic agents. For example, a compound oragent that targets CTGF may sensitize the tumor to the action of asecond therapeutic agent and be used to limit or reduce tumor cellsurvivability, i.e., provide a cytostatic effect; or may reduce tumormass, i.e., provide a selective cytoxic effect; while a traditionalchemotherapeutic or radiation may be used to reduce tumor mass, i.e.,provide a non-specific cytotoxic effect. Use of the present methods inthis context would allow the use of smaller doses of chemotherapeutic orradiation to achieve the desired end result, thus reducing adverse sideeffects commonly associated with traditional non-specific cancertherapies. Additionally, the compound or agent may be combined with asecond therapeutic agent, e.g., an anti-angiogenic agent such asAvastin; an anti-receptor tyrosine and/or serine/threonine kinase agentsuch as Tarceva, etc.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

EXAMPLES

The invention will be further understood by reference to the followingexamples, which are intended to be purely exemplary of the invention.These examples are provided solely to illustrate the claimed invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods that are functionally equivalent arewithin the scope of the invention. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Example 1 Generation of Cell Lines Expressing Different Levels of CTGF

MIA PaCa-2 cells (American Type Culture Collection (ATCC), Manassas Va.)are reportedly insensitive to the growth inhibitory effects of TGF-β1.(See, e.g., Freeman et al. (1995) J Cell Physiol 165:155-163.) Todetermine if MIA PaCa-2 cells would provide a suitable host forexongenous CTGF expression, both constitutive and TGFβ-inducibleexpression of CTGF was determined by measuring CTGF levels in cellculture supernatants using an ELISA assay that measures both cleavedN-terminal fragment and whole CTGF. (See International Publication No.WO 03/024308, incorporated by reference herein in its entirety.) As CTGFexpression was not detected in either untreated or TGF-β2-treatedcultures, MIA PaCa-2 cells were used to generate cells expressingvarious levels of CTGF.

MIA PaCa-2 cells were transfected with pSCMV-Puro-CTGF, an adenoviralconstruct encoding full-length CTGF and the puromycinantibiotic-resistance gene, using lipofectamine 2000 reagent (InvitrogenLife Technologies, Carlsbad Calif.) according to procedures supplied bythe manufacturer. Individual clones were selected based on theirresistance to puromycin, and CTGF expression and protein levels weredetermined by quantitative RT-PCR and ELISA assay, respectively. Aseries of clones were isolated and characterized, and representativeclones that exhibited low (clones CB1 and CB2), medium (clones CE8 andCD2), and high (clones CA9 and CB4) levels of CTGF expression andprotein production were identified. On plating 10⁵ cells, the low-,medium-, and high-CTGF expressing clones showed clear differences inCTGF expression as determined by quantitative RT-PCR, with secretedlevels of CTGF ranging from a low of about 0.3 to a high of about 2.2 μgCTGF/48 hrs. (FIG. 1.)

MIA PaCa-2 cells transfected with vector encoding the puromycinresistance gene, but not with CTGF, served as control (vector). As wasseen in the MIA PaCa-2 parental cell line, cells transfected withcontrol vector did not express detectable levels of CTGF transcript orprotein.

Example 2 CTGF Enhances Anchorage-independent Growth

In vitro cellular growth rates of vector control and low-, medium-, andhigh-CTGF expressing clones were measured to determine whether higherlevels of CTGF expression alter the ability of tumor cells to surviveand/or proliferate. Clonal cell lines were cultured in standard cultureplates for up to 12 days, with cells harvested and counted after 3, 6, 9and 12 days. No difference in the rate or level of cell accumulation wasseen between vector control cells and cells expressing different levelsof CTGF (FIG. 2A).

AIG is a common characteristic of cancer cells. Although CTGF apparentlywas unable to induce AIG independently, it was required for theinduction of AIG by TGF-β1. (See, e.g., Frazier et al. (1996) J InvestDermatol 107:404-411; and Kothapalli et al. (1997) Cell Growth Differ8:61-68.) MIA PaCa-2 cells reportedly have a colony-forming efficiencyin soft agar of approximately 19%. (Yunis et al. (1977) Int J Cancer19:128-135.) To determine the effect of CTGF expression on AIG in MIAPaCa-2 cells, clones expressing various levels of CTGF (CA9, CB1, CB4,and CD2) or containing vector alone (VA2, VA6, VB1, and VB4) wereanalyzed for their ability to grow in soft agar.

In duplicate experiments, approximately 1.2×10³ cells from each clonewere distributed in 2.0 ml of 0.35% Noble Agar containing 10% fetalbovine serum (FBS) and 10% newborn calf serum (NCS). Each embedded cellmixture was overlaid on 1.5 ml of 0.7% Noble Agar in 6-well plates, anda 1.5 ml top layer of 0.7% Noble Agar was added to each well to preventevaporation. Plates were incubated for 11 days in a humidified incubatorat 37° C., 5.0% CO₂. The number of colonies was enumerated by counting a1.5 cm-by-1.5 cm grid under a microscope. Total colony counts wereextrapolated to the entire plate based on the ratio of the surface areaof each well to the surface area of the grid. Colony morphologies werephotographed at 20× magnification.

Although MIA PaCa-2 cells transformed with vector alone demonstratedAIG, the number of colonies formed increased with increasing CTGFexpression. (FIG. 2B.) The top panels in FIG. 3A show colony morphologyof CTGF-expressing clones and null vector clones, respectively. Morecolonies formed in CTGF-expressing clones, and colonies arising fromCTGF-expressing clones were generally larger than colonies arising fromnull vector clones. These results demonstrate that CTGF enhancesanchorage-independent growth of MIA PaCa-2 cells, and higher levels ofCTGF expression result in larger colony size.

Example 3 Agents Inhibiting CTGF Reduce Anchorage-independent Growth

As CTGF enhances AIG (see above example), agents that reduce CTGFactivity and/or availability were tested in the AIG assay to determineif they could reduce colony formation. The ability of antibodies thatspecifically target CTGF, such as those generally described in U.S. Pat.No. 5,408,040, to inhibit AIG in MIA PaCa-2 cells was tested using thesoft agar assay as described above with the following modifications. Ahuman monoclonal antibody, CLN-1 (see International Publication WO2004/108764), which specifically binds CTGF, or a control human IgG wasadded at a final concentration of 100 μg/ml to soft agar containingCTGF-expressing clone CD2 cells or null vector clone VA6 cells. Plateswere re-fed on day 5 with 1.5 ml top layer containing 100/g/ml of CLN-1or human IgG. Plates were incubated for 11 days and assessments werecarried out as described above.

As shown visually in the lower panels of FIG. 3A, CLN-1 treatmentsignificantly inhibited colony formation by the CTGF-expressing cells.Quantification of the result shows that antibody reduced colonyformation by CTGF-expressing cells approximately 85%, but produced onlya weak inhibition in colony formation by the vector clones. (FIG. 3B.)Control IgG had no effect on basal colony formation by either vectorcontrol or CTGF-expressing cells. The results demonstrate thattherapeutics targeting CTGF activity and/or availability are effectivein reducing or inhibiting anchorage independent growth in MIA PaCa-2pancreatic cancer cells and potentially in cancer cells generally.

Example 4 CTGF Enhances Tumor Expansion and Increases Host Mortality

Female 8 to 10 week old SCID (severe combined immune deficient; SimonsenLaboratories, Inc., Gilroy Calif.) or athymic nude (nu/nu; Harlan,Indianapolis Ind.) mice were injected subcutaneously with approximately10⁷ cells from control, low-, medium-, or high-CTGF expressing MIAPaCa-2 clones. Injected mice were monitored for tumor expansion, withtumor size and volume (calculated as length×width×height) measured atweekly intervals. Measurements for animals receiving injection of thesame clone type were averaged. Tumors were excised and embedded inparaffin. Sequential 4 μm paraffin sections were stained with rabbitantibody (1:50; Zymed Laboratories, Inc., South San Francisco Calif.)directed against the Ki-67 nuclear antigen, which is only present inproliferating cells (Gerdes et al. (1984) J Immunol 133:1710-1715), toquantify proliferative cells. Detection was performed using biotinylatedsecondary antibodies in combination with horseradish-peroxidase-coupledstreptavidin (Jackson ImmunoResearch Laboratories, Inc., West Grove Pa.)and diaminonobenzidine substrate (Invitrogen). Separate sections werestained by terminal deoxynucleotidyl transferase biotin-dUTP nick endlabeling (TUNEL), which identifies apoptotic cells, using the DEADENDfluorometric TUNEL system (Promega, Madison Wis.) according to themanufacturer's instructions. Briefly, sections were deparaffinized andrehydrated, permeabilized in proteinase K, and treated with terminaldeoxynucleotidyl transferase incubation buffer at 37° C. for 60 minutesin the dark. Sections were counterstained with4′-6-Diamidino-2-phenylindole (DAPI, Sigma).

The percentage of proliferating (Ki-67-positive) and apoptotic (TUNELpositive) tumor cells was determined in four randomly selected areas oftumor sections using a Nikon Eclipse E800 microscope at 400×magnification. At least 400 cells per high power field were counted todetermine the percentage of positive cells. Mean values and standarddeviations were calculated.

Mice injected with CTGF-expressing clones exhibited enhancedtumorigenesis proportional to the level of CTGF expression, asexemplified by increase in tumor volume over time in animals injectedwith control, medium, and high expressing cells. (FIG. 4A.) Miceimplanted with control MIA PaCa-2 cells showed essentially no tumorexpansion. (FIG. 4A.) Furthermore, increased expression of CTGF in cellimplants correlated with increased mortality in injected animals; FIG.4B shows a Kaplan-Meier Cumulative Survival Plot through time to lastfollow up or time of death. CTGF-expressing tumors also showed increasednumbers of actively proliferating cells (FIG. 5A) and reduced numbers ofactively apoptotic cells (FIG. 5B) within the tumor. Although CTGF wasoriginally described as a mitogenic factor (see, e.g., Bradham et al.(1991) J Cell Biol 114:1285-1294), the increased cell proliferation maybe directly or indirectly induced by CTGF. These results demonstratethat CTGF is directly involved in tumor expansion, and is thereby atarget for therapeutic agents that may reduce or inhibit tumor expansionin vivo. In particular, agents that block or neutralize CTGF functionmay provide therapeutic benefit in tumor-bearing patients.

Example 5 Plasma and Urine Levels of CTGF

As CTGF is a secreted factor, increased production of CTGF by anexpanding tumor mass would lead to increased CTGF in the surroundingstroma and, potentially, throughout the circulation of the host. Todetermine whether tumor-bearing hosts had higher circulating levels ofCTGF, urine and plasma samples were collected from mice followinginjection of MIA PaCa-2 clones and expansion of detectable tumor mass.Plasma samples were also obtained by terminal bleed. CTGF levels weremeasured using the ELISA assay described in Example 1. (SeeInternational Publication No. WO 03/024308.) CTGF expression levels werealso reassessed in culture supernatants from control clones or thoseexpressing low, medium or high levels of CTGF.

The level of CTGF present in both plasma (FIG. 6B) and urine (FIG. 6C)of tumor-bearing hosts mimicked CTGF expression observed in vitro inculture supernatants of the respective MIA PaCa-2 clones (FIG. 6A).Thus, mice implanted with high CTGF expressing clones exhibited thehighest levels of circulating and excreted CTGF, and animals implantedwith low CTGF expressing cells exhibited the lowest detectable levels ofcirculating and excreted CTGF. The levels of circulating and urinaryCTGF levels in animals injected with control cells were undetectable.The data demonstrate a direct correlation between CTGF expression levelsin the tumor and CTGF levels found in body fluids, suggesting tumorssecrete CTGF directly into their environment.

Since the degree of CTGF expression in tumor cells may directlycorrelate with increased tumor expansion and survival, and the level ofCTGF produced by a tumor is proportional to the level of CTGF in bodyfluids such as blood and urine, measurement of CTGF in, e.g., plasma orurine of cancer patients may be indicative of tumor stage or correlatewith tumor burden, which would be a valuable diagnostic and prognosticbiomarker for disease assessment.

Example 6 Agents Inhibiting CTGF Reduce Tumor Expansion 6.1 SubcutaneousImplantation of CTGF-expressing MIA PaCa-2

In two separate studies, mice were implanted subcutaneously with eithermedium or high CTGF expressing MIA PaCa-2 clones and tumor size andvolume were determined in each set of animals at weekly intervals. Whentumors reached a volume of approximately 250 mm³, animals werestratified to receive either 20 mg CLN-1 per kg body weight or phosphatebuffered saline (antibody vehicle) twice a week. Measurement of tumorsize and volume was continued for each group of animals at weeklyintervals.

Administration of antibody to nude mice implanted with high CTGFexpressing clones resulted in slowed tumor expansion, particularlyapparent after approximately two weeks of therapy. (FIG. 7A.) Similarly,administration of antibody to animals implanted with medium CTGFexpressing clones resulted in reduced tumor expansion as compared toanimals receiving vehicle control. The data show that administration ofan agent targeting CTGF, e.g., a CTGF-specific monoclonal antibody,leads to reduction in tumor expansion shortly after initiation oftherapeutic administration, e.g., after one to two weeks of treatment.

Therapeutic agents targeting CTGF exert benefits at various stages oftumor progression. For example, high-CTGF expressing cells weresubcutaneously injected into mice to generate xenograft tumors asdescribed above. In one cohort, mice were administered 20 mg/kg CLN-1twice a week beginning approximately two days after injection of cells.In a second cohort, tumors were allowed to expand to a volume ofapproximately 250 mm³, at which time the cohort was stratified andeither left untreated or treated with 20 mg/kg antibody twice a week.Measurement of tumor size and volume was continued for each group ofanimals at weekly intervals. In both cohorts, administration of antibodyreduced tumor expansion.

In a similar study, high CTGF expressing cells were subcutaneouslyinjected into mice to generate xenograft tumors as described above, withone cohort of mice left untreated and one cohort administered 20 mg/kgantibody therapy twice a week beginning approximately two days aftertumor implantation. Consistent with results shown above, administrationof antibody inhibited tumor expansion and progression, such that by 8weeks after implantation the vehicle control treated mice had averagetumor volumes approximately twice that of the antibody-treated cohort.However, termination of antibody therapy at 8 weeks resulted inresumption of tumor expansion.

The data show that administration of therapeutic agents targeting CTGFreduces or prevents tumor expansion at various stages of tumorprogression. Thus, agents that block CTGF may provide therapeuticbenefit in cancer patients that present with either early stage orclinically established tumors. The results demonstrate that agentstargeting CTGF may provide benefit to cancer patients at various stagesof disease, retarding expansion of early and late stage tumors. Suchtherapy may be particularly advantageous in combination with a secondtherapeutic method that exerts a cytotoxic effect, e.g., traditionalchemotherapy or radiation therapy. The CTGF-directed therapeutic wouldeffectively prevent tumor expansion, while the cytotoxic therapydestroyed the existing tumor mass.

6.2 Subcutaneous Implantation of PANC-1

Nude mice (Harlan) were implanted subcutaneously with PANC-1 cells in aprocedure analogous to the one described in Example 6.1. Mice injectedwith PANC-1, which express CTGF endogenously, exhibited an increase intumor volume over time. (FIG. 7B.) As was seen with tumors derived fromCTGF-expressing MIA PaCa-2 cells, administration of antibody CLN-1 toanimals implanted with PANC-1 resulted in slowed tumor expansion. (FIG.7B.) Again the data show that administration of therapeutic agentstargeting CTGF reduce or prevent tumor expansion.

Analysis of tumors for proliferating and apoptotic cells, using methodsdescribed above, showed that agents targeting CTGF do not significantlyaffect the number of proliferating cells in the tumor (FIG. 8A), but dorestore apoptotic activity of cells within the tumor (FIG. 8B). Theseresults suggest that CTGF is important for survival of proliferatingcells, thus increasing cell mass by maintaining the viability ofproliferating cells and reducing cell apoptosis. The results furtherdemonstrate that administration of an agent that targets CTGF providestherapeutic benefit.

6.3 Orthotopic Implantation of PANC-1

Tumors were initiated by subcutaneous injection of approximately 10⁶PANC-1 cells into the flank of nude mice (Harlan). After 2-6 weeks thecells formed primary tumors that were aseptically resected, immediatelyminced into 2-mm³ pieces, and implanted into the pancreas of naïve nudemice via a surgical flap. Mice were randomized to receive eitherphosphate buffered saline (vehicle control; n=4) or 20 mg/kg CLN-1administered by intraperitoneal injection, with dosing initiated 2 weeksafter tumor fragment implantation and dosed twice weekly thereafter.Treatment was continued for a total of 6 weeks, at which time controlmice had developed large abdominal tumor masses, and all the mice weresacrificed. Primary tumor volumes were calculated using the equation:VOLUME=(L×H×W)×π/4, where L, H, and W are the length, height, and widthof the tumor.

Compared to tumor-bearing animals receiving only vehicle control,animals administered CTGF-specific antibody produced tumorsapproximately 83% smaller, presumably due to reduced rate of tumor cellsurvivability. (FIG. 9.) The result shows that antibodies specific forCTGF reduce the expansion of orthotopic pancreatic tumors in vivo.Additional data using an anti-CTGF antibody that binds host-derivedmouse CTGF, but not PANC-1-derived human CTGF, suggest CTGF derived fromboth the tumor and surrounding normal tissue may be involved in tumorexpansion. Thus, similar to results presented above, the presentexperiment demonstrates that agents targeting CTGF may providetherapeutic benefit in treating patients with pancreatic cancer byreducing or preventing tumor expansion and/or progression.

Example 7 Agents Inhibiting CTGF Reduce Tumor Metastasis

Experimental animals that were treated as described in Examples 6.2 wereassessed visually at autopsy for any evidence of tumor metastases toaxillary and inguinal lymph nodes. Table 1 shows lymph node metastasesin animals treated with CLN-1 relative to animals treated with vehiclecontrol. As shown in the table, 5 out of 6 tumors metastasized to lymphnodes in control animals, whereas only 1 out of 5 tumors metastasized inantibody-treated animals.

TABLE 1 Control antibody CLN-1 Macroscopic Macroscopic LN metastasis LNmetastasis Tumor # Axillary Inguinal Tumor # Axillary Inguinal 1 − +1 + + 2 + + 2 − − 3 + + 3 − − 4 + − 4 − − 5 + + 5 − − 6 − −

Experimental animals that were treated as described in Examples 6.3 wereassessed visually at autopsy for any evidence of tumor metastases toproximal and distal lymph node and other sites within the peritoneum.Primary tumor volume and sites of metastases were further analyzed anddetails for each animal are provided in FIG. 9. In the figure, symbolsrepresent tumor volume, and letters beside the symbol indicate sites ofmetastasis as follows: M=mesenteric, G=gastric, P=peritoneal, andA=ascites.

The results demonstrate that therapeutics targeting CTGF not only reducetumor expansion and survivability, but significantly reduce metastasisof the primary tumor to secondary sites. Therefore, agents targetingCTGF may provide therapeutic benefit in treating patients with cancer,particularly pancreatic cancer, by reducing tumor expansion and reducingor preventing metastasis of existing tumors.

Example 8 Agents Inhibiting CTGF Affect Tumor Microvessel Formation

Tumors derived from the experimental animals described in Example 6.3(supra) were embedded and frozen in OCT compound prior to beingsectioned and stained with a rat anti-mouse CD31 monoclonal antibody andcounterstained with hematoxylin. A total of three angiogenic hot-spotsper slide were analyzed and scored for blood vessel number, andquantitated for microvessel density using an Image Pro Plus version4.5.1 image analysis program (Media Cybernetics, Silver Spring, Md.).Microvessel density was calculated as the ratio of positively stainedareas to the total area of the image or field.

As compared to tumor-bearing animals that received vehicle control, thenumber and density of tumor blood vessels was reduced by 45% and 61%,respectively, in animals receiving CLN-1. The results indicate thatagents targeting CTGF reduce the number and density of blood vesselsthat form in primary tumors, particularly pancreatic tumors. A reductionin vascularization may be one mechanism by which CTGF limits tumorexpansion, and provides further support for the use of agents targetingCTGF for treatment of various cancers, particularly highly vascularizedcancers.

The present invention demonstrates that therapeutic agents targetingCTGF are effective at preventing or reducing tumor expansion, anddecreasing the number and density of microvessels within tumors. Theinvention further demonstrates that therapeutic agents targeting CTGFare additionally effective at reducing and/or preventing metastasis ofthe primary tumor to the lymphatic system and/or other organs. Together,the data indicate that agents targeting CTGF may provide therapeuticbenefit in pancreatic and other neoplastic disease by inhibiting thecapacity of tumors to expand, survive, and metastasize to other siteswithin the body.

Various modifications of the invention, in addition to those shown anddescribed herein, will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims.

All references cited herein are hereby incorporated by reference intheir entirety.

1. A method for treating pancreatic cancer in a subject, the methodcomprising administering to the subject a therapeutically effectiveamount of an agent that reduces the expression or activity of connectivetissue growth factor (CTGF) such that the metastasis of the pancreatictumor is reduced or prevented, thereby treating the cancer in thesubject.
 2. The method of claim 1, wherein the pancreatic tumor is anadenocarcinoma.
 3. The method of claim 1, wherein the pancreatic tumoris a ductal adenocarcinoma.
 4. The method of claim 1, wherein the agentis an oligonucleotide that specifically binds to a polynucleotideencoding CTGF and reduces CTGF expression.
 5. The method of claim 4,wherein the oligonucleotide is selected from the group consisting of aninterfering ribonucleic acid (siRNA), a micro-RNA (miRNA), and anantisense sequence.
 6. The method of claim 1, wherein the agent is anantibody that specifically binds to CTGF.
 7. The method of claim 6,wherein the antibody is a monoclonal antibody or a fragment thereof thatspecifically binds to CTGF.
 8. The method of claim 6, wherein theantibody is CLN-1 or a derivative thereof that specifically binds toCTGF.
 9. The method of claim 1, wherein the method additionallycomprises administering to the subject a therapeutically effectiveamount of a cytotoxic chemotherapeutic compound.